New Diterpenoids from the Aerial Parts of Salvia reuterana.

The genus Salvia is a valuable origin of structurally diverse terpenoids. In a project directed at structurally interesting bioactive metabolites from Iranian Salvia species, we studied Salvia reuterana. Two new labdane diterpene, 6β, 14α-dihydroxy-15-acetoxysclareol (1), and 14α, 15- dihydroxy sclareol (2), were isolated from the aerial part of the plant. Their structures were established mainly by 1D and 2D NMR spectroscopic techniques, including 1H-1H COSY, HSQC and HMBC methods and HR-ESI-TOFMS spectral data. Compound 1 and 2 were tested for their inhibitory activity toward HeLa and MCF-7 cell lines. Our results showed that S. reuterana is a rich source of labdane diterpenoids. These compounds are rather rare in Salvia species, although they are frequently found in other genera of the Lamiaceae. S. reuterana is a new source of these diterpenoids.


Introduction
Salvia, consisting of about 1000 species, is the largest genus in the family Lamiaceae and widely distributed in various regions of the world, Namely, the Mediterranean area, south Africa, central and south America, and southeast Asia (1). Plant of the genus Salvia have attracted much attention owing a variety of medicinal properties and biological activities, such as antibacterial, antioxidant, to antitumor, antidiabetic and anti-Inflammatory activities (1-3). The Iranian flora comprises 61Salvia species, 17of which are endemic (4). Some Iranian Salvia species have been investigated from a phytochemical viewpoint, and antibacterial, antiprotozoal, and anticancer compounds have been reported (5-12.). Salvia reuterana Boiss.
is an endemic species which grows in the highlands of central Iran (13). Its common name in Persian is "Mariam Goli Esfahani" (14), and the aerial parts of the plant are traditionally used as sedative and anxiolytic herbal medicine. In addition, the antibacterial, antioxidant, free radical scavenging and anti-anxiety properties of this herb have been proved in recent studies (15)(16)(17)(18)(19). In a previous work, we have reported the presence of six labdane diterpenoids from this plant (20). Further examination of the n-hexane extract of S. reuterana led to the isolation of two new other labdane diterpenoids (1, 2).

Experimental
General HR-ESI-MS was carried out on a Bruker 147 microTOF-ESIMS system. NMR spectra were recorded on Bruker AVANCE III-500 spectrometer operating at 500.13 MHz for 1 H NMR and 125.77 MHz for 13 C NMR with TMS as an internal standard. Silica gel (70-230 and 230-400mesh) used for column chromatography and silica gel F 254 (20×20 cm) for TLC, were both supplied by the Merck company.

Plant material
The aerial parts of Salvia reuterana were collected from the northern hilly areas of Tehran in Juley 2011. A voucher specimen (MPH-1321) has been deposited in the herbarium of Medicinal Plants and Drugs Research Institute (MPH) of Shahid Behshti University, Tehran, Iran.

Extraction and Isolation
The air-dried aerial parts of S. reuterana (3.0 kg) was crushed and extracted with n-hexane (3 × 15 L) by maceration at room temperature. Extract was concentrated in vacuo, to afford 130g of a dark gummy residue. The residue was separated on a silica gel column (230-400 mesh, 850g) with a gradient of n-hexane-EtOAc (100/0 to 0/100) as eluent, followed by increasing concentration of MeOH (up to 25%) in EtOAc. On the basis of TLC analysis, fractions with similar composition were pooled to yield 27 combined fractions.

Cytotoxicity assay
The human epithelioid cervix carcinoma (Hela) and human breast adenocarcinoma (MCF-7) cell lines were purchased from National Cell Bank of Iran (NCBI), Pasteur Institute of Iran (Tehran, Iran), and maintained in DMEM medium supplemented with 10% fetal bovine serum and 100 U/mL penicillin and 100 μg/mL streptomycin. These cells were kept at 37 °C in a humidified atmosphere containing 5% CO 2 . Compounds 1-3 were dissolved in DMSO to make a stock of 1mg/mL and further diluted to final concentrations of 10-100 μg/mL with the serum free culture medium.

Cell viability assay
Cell viability was determined using the MTT assay. Briefly, 2.5×104 cells were seeded in 96-well plates at 37 °C with 5% CO 2 for overnight incubation and treated with appropriate concentrations of compounds 1-3 for 24 h. The cells were then incubated with a serum-free medium containing MTT at a final concentration of 0.5 mg/mL for 4 h. The dark formazan crystals formed were dissolved in DMSO and the absorbance was measured at 570 nm.

Results and Discussion
Compound 1 was obtained as a white amorphous powder. Its HI-ESI-TOFMS exhibited a molecular ion peak at m/z 423.2738 [M + Na + ], agreed with the molecular formula C 22 H 40 O 6 . The molecular formula accounted for three degrees of unsaturation. The 13 C NMR spectrum (Table 1), showed 22 carbon resonances which were analyzed from the DEPT-HSQC spectra to consist of six methyls, seven methylens, four methines and four quaternary carbons. Two carbon signals at δ C 73.1 and 73.4 indicated the presence of oxygen bearing sp 3 quaternary carbons. Another oxygenated carbons at δ C 75.1 and 67.6 accounted for the carbons carrying two hydroxyl groups with the carbinolic methine protons coming at δ H 3.55 (1H, dd, J = 2.5, 8.5 Hz) and δ H 4.39 (1H, brd, J= 2.6 Hz), respectively. The signal at δ C 66.2 the Aerial Parts of Salvia reuterana accounted for amethylenic carbon attached to an acetoxy group, with the corresponding methylene protons coming at 3.96, 4.22 each as a doublet of doublet. According to the degree of unsaturation the molecule was bicyclic and appeared to be a labdane diterpenoid (21). The 1 H and 13 C NMR spectrum displayed features similar to those of 14α-hydroxy-15-acetoxysclareol, isolated previously from this plant by us (20). However, the 13 C NMR spectrum of 1 showed the presence of an additional methine at δ C 67.6 instead of the methylene group (C-6). Hence, the methylene was replaced by an oxygenated methine. The signals of C-7 (δ C 51.3) and C-5 (δ C 56.7) were paramagnetically shifted (Δδ +7.3 and +0.7 ppm, respectively) in comparison to those of 14α-hydroxy-15-acetoxysclareol. Also the resonances of neighboring H-7α (δ H 1.55), H-7 (δ H 1.91), and H-5α (δ H 0.83) were shifted (Δδ= +0.2, +0.15, and -0.03ppm, respectively). HMBC correlations between H-5α, H-7α with C-6, and between H-6, C-8, and C-10 ( Figure  1) confirmed the location of the hydroxyl group. Diagnostic COSY correlations were observed between H-6 and H-7αand H-7β, and between H-6 and H-5. The relative configuration of the hydroxyl group at C-6 was determined as β based on the magnitude of the vicinal coupling constants of the H-6 resonance (δ H 4.39, brd, J = 2.6 Hz). NOESY contacts of H-6 with H-5 and H-7ax were observed. The 1 H chemical shifts of CH 3 -17, CH 3 -19 and CH 3 -20 (δ H 1.32, 1.10 and 1.08, respectively) appeared downfield (δ H 0.21, 0.38, and 0.35) relative to those of 14α-hydroxy-15-acetoxysclareol. These differences were in agreement with an axial orientation of the    hydroxyl group at C-6. Thus, the structure of 1 was established as 6β,14α-dihydroxy-15acetoxysclareol.
Compound 2 was obtained as a white powder. The HR-ESI-TOFMS showed a peak at m/z 365.2712 [M+Na] + , in agreement with the elemental formula C 20 H 33 O 4 , which accounted for three degrees of unsaturation. The 1 H and 13 C NMR data (Table 1) strongly resembled those of 1, indicating that the two compounds were structurally related. Inspection of the 1 H and 13 C NMR spectra showed the lack of the signals belonges to the acetoxy group in compound 2. Also, the methine signals at67.6 ppm (δ H 4.39) was replaced by a methylene group (δ C 20.6 and δ H 1.57, 1.18) in 2. Thus, compound 2 was established as 14α, 15-dihydroxy sclareol.
Compounds 1 and 2 were evaluated for their in vitro cytotoxic activity against Hela (human epitheloid cervix carcinoma) and MCF-7 (human breast adeno-carcinoma) cell lines. The results of the cytotoxicity studies were indicated in Table 2.

Conclusion
Our results showed that S. reuterana is a rich source of labdane diterpenoids. The most abundant diterpenoids in the genus are abietanes and rearranged abietanes. Labdane diterpenoids are rather rare in Salvia species although they are frequently found in other genera of the Lamiaceae. S. reuterana is a new source of these diterpenoids. (2)